Dr. Sangpil Yoon is a postdoctoral scholar at the Department of Biomedical Engineering at the University of Southern California (USC). He has developed an innovative technique for the intracellular delivery of macromolecules using high frequency ultrasound. Dr. Yoon is a perfect candidate for NIH Pathway to Independence Award (K99/R00) and he will smoothly transit from a mentored trainee to an independent investigator by completing research goals and career development training plans, proposed in this award proposal. Dr. Yoon has three mentors to provide their expertise and constructive and critical advice during his award period. Dr. Yoon will have trainings in ultrasound and medical ultrasonic transducer development from Dr. Kirk Shung, USC, molecular and cellular biology from Dr. Yingxiao Wang, University of California, San Diego (UCSD), and stem cell and regenerative medicine from Dr. Qi-Long Ying, USC. Transfection methods currently available in research laboratories and clinics are based on non-targeted and random process such as viral-vectors and electroporation. I have developed an innovative transfection method by focusing acoustic energy within very confined area of less than 10 ?m using a very high frequency ultrasonic transducer with a center frequency of over 150 MHz, entitled acoustic-transfection. The key innovation is that the acoustic-transfection has the capabilities of single-cell level targeting and controlling the size of delivered macromolecules with low cytotoxicity. The hypothesis is that the developed acoustic- transfection can deliver various kinds of macromolecules into different cells with the peculiar capabilities that distinguish from other transfection methods. Understanding of signaling pathways and the activation of important molecular events during intracellular and intercellular interactions are important because these are a basic building block to identify cell phenotypes and some molecular events are precedent for certain disease. Based on this understanding, engineering cell fate and cell functions using efficient gene editing with CRISPR/Cas9 and the generation of iPSCs by delivering recombinant proteins for regenerative medicine will further advance human health. Therefore, the primary goal of this proposal is to deliver desired molecules using acoustic-transfection into designated cells to visualize cell-to-cell interactions and the signaling of important molecular events, to induce gene expressions, and to demonstrate the delivery of recombinant proteins labeled with fluorescence dyes. To test hypothesis and to achieve the goal of this proposal, three specific aims were developed. 1) I will further optimize acoustic- transfection using macromolecules with different sizes. 2) I will deliver FRET biosensors into neighboring or single cells to visualize molecular events under stimulation. 3) pCas9_GFP will be delivered into hESCs and GFP expression will be observed. Recombinant proteins conjugated with Alexa 488 will be continuously delivered into hNSCs using acoustic-transfection for 8 days to confirm short-term delivery efficacy and long- term endogenous pluripotency. The proposed acoustic-transfection will advance transfection methods with the safe delivery of versatile molecules into cells to better understand important molecular events in cells and develop improved therapeutic strategies to cure diseases.